Biparatopic antibody BA7208/7125 effectively neutralizes SARS-CoV-2 variants including Omicron BA.1-BA.5

SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1–BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.

References for the additional methods 23 Figure S1 to S10 24

Binding free energy calculation 28
To calibrate the binding affinities of the S protein with antibodies and predict the 29 binding ability of the BA7054-7125-RBD complexes which lacked the experimental 30 data, we used the Flex ddG protocol 1 implemented within Rosetta to calculate the 31 changes in binding free energies (interface ∆∆G) upon mutation. All Spikes were 32 truncated to include only the RBD domain in the S protein  The data from the Delta Spike RBD (monomer)-antibody were set as the references, 34 with which the values of all other variants were compared. The ∆∆G calculation of 35 each system was repeated 50 times and the average values with two different 36 scoring types were reported. 37 38 Cryo-EM specimen preparation and data acquisition for the Omicron S-BA7208 39 complex 40 90 μL purified Omicron S protein at the concentration of 1.37 mg/mL was incubated 41 with 10.1 μL BA7208 Fab at the concentration of 3.99 mg/mL at a 1:3 molar ratio on 42 ice for 40 min for the next step of size exclusion chromatography (SEC). After 43 centrifugation (16200 g, 4°C for 5 min), 100 μL supernatant was injected to GE micro 44 Akta using Superose 6 column. The peak fraction was applied for cryo-EM grid 45 preparation. 46 An aliquot of 4 μL protein sample of Omicron S-BA7208 complex was applied 47 onto a glow-discharged 300 mesh grid (Quantifoil Au R1.2/1.3) supported with a thin 48 layer of GO (Graphene Oxide), blotted with filter paper for 3.0 s and plunge-frozen in 49 liquid ethane using a Thermo Fisher Vitrobot Mark IV. Cryo-EM micrographs were 50 collected on a 300kV Thermo Fisher Titan Krios G3i electron microscope equipped 51 with a K3 direct detection camera and a BioQuantum image filter (GIF: a slit width of 52 20eV). The micrographs were collected at a calibrated magnification of x81,000, 53 yielding a pixel size of 0.856 Å at a super-resolution mode. In total, 2,554 54 micrographs were collected at an accumulated electron dose of 50e -Å -2 s -1 on each 55 3 micrograph that was fractionated into a stack of 32 frames with a defocus range of -56 1.0 μm to −2.0 μm. 57 58 EM data processing 59 Beam-induced motion correction was performed on the stack of frames using 60 MotionCorr2 2 . The contrast transfer function (CTF) parameters were determined by 61 CTFFIND4 3 . A total 2,554 good micrographs were selected for further data 62 processing using Relion 3.1 program 4 . Particles were auto-picked by the Auto-picking 63 program in Relion, followed by two rounds of reference-free 2D classifications. Next, 64 633,788 particles were selected from good 2D classes and were subjected to two 65 rounds of 3D classification using a reconstruction of the Omicron S-BA7208 complex 66 as a starting model. Two converged 3D classes with a feature contains one Omicron 67 S-BA7208, one Omicron S were selected for a final round of 3D refinement. In the 68 two 3D classes, 495,537 particles from a 3D class showing the highest resolution 69 feature with an additional density were selected for a round of 3D refinement, yielding

Model building and refinement 75
The model of Omicron S-BA7208 complex was built by fitting the model of structure 76 of Omicron S-BA7208 (predicted by AlphaFold2) into the density map using UCSF 77 Chimera 5,6 , followed by a manual model building of Omicron S-BA7208 complex 78 molecules in COOT 7 and a real space refinement in PHENIX 8 . The model statistics 79 were listed in Supplementary information,

Fig. S1 Graphical Abstract for antibody identification and characterization
mAbs against SARS-CoV-2 variants were produced through sequential immunization and followed sequential screening from human antibody transgenic mice. ScFvs of potential clones were sequenced and then converted into IgG format for expression.
Antibodies showing RBD/ACE2 receptor blocking activity were obtained for in vitro neutralization activity evaluation. Structure analysis was performed for the broadly potent neutralizing mAbs.  617.2, B.1.1.529.1 and B.1.621 Table S1. Binding free energy calculation. The systems with relatively higher binding free energies than the Delta system are highlighted in blue, and those with lower ones are highlighted in orange. The BA7208 shows good binding ability for all kinds of Spike RBD. While the BA7054 and BA7125 show weaker bindings for Omicron Spike RBD, especially in the BA7125-RBD systems. These results are consistent with the experimental data we observed. Of note is the BA7054-7125-RBD systems, where the binding ability for Omicron is not as good as other variants.  Disallowed (%) 0.00 Table S3. Protein-protein interaction between Delta RBD and BA7208 Fab.

Hydrogen bond
Thr ( Table S4. Protein-protein interaction between Delta RBD and BA7125 Fab.

Hydrogen bond
Arg (

Hydrogen bond
Arg (

Residue in
Omicron RBD

Hydrogen bond
Thr (